Transform biomass into fuel takes work. In the case of sugarcane, main raw material studied for this purpose in Brazil, processes are needed to break the lignocellulosic biomass into simple fermentable sugars. These operations are performed by sophisticated enzymatic complexes, obtained from various types of microorganisms. When everything goes right we still need to handle about 20-35% of the formed polymers that tend to be rejected by industrial yeasts, drastically reducing the profitability of ethanol produced.

The solution to such biotechnological dilemmas may lie in the rich Brazilian biodiversity. Researchers at the National Center for Research in Energy and Materials (CNPEM) found in the intestinal tract of the chrysomelid beetle larvae, a new yeast species of the genus Pseudozyma capable to metabolize five-carbon sugars and to secrete an enzyme of interest, xylanase, in large quantities.

The class of enzyme produced by Pseudozyma brasiliensis sp.nov, proposed name for the new species has a high biotechnological potential. It can be used in the fiber degradation of sugarcane bagasse for the conversion of second generation ethanol as well as in the paper, food and feed industry. It also serves the production of xylitol and xylooligosaccharides.

Juliana Velasco de Oliveira, a researcher at the the National Laboratory of Science and Technology of Bioethanol (CTBE) – belonging to CNPEM – explains that the xylanolytic activityof the P. brasiliensis was about 20 times higher than the Aspergillus niger, fungus notoriously recognized by the expression of this type of enzyme. “The xylanase of this yeast, called PbXynA, has a specific activity greater than that of any other similar activity already described,” says Oliveira. This may represent a considerable advance in one of the main technological barriers in the production of second generation ethanol that is building effective enzyme cocktails in the degradation of the biomass.

Genome sequencing of P. brasiliensis yeast, identified at the CTBE.

The study which led to the identification of a new yeast species was started two years ago Seven different types of insects that act as pests of sugarcane (chrysomelid, pão de galinha, migdolus, among others) were collected in the plantations of Ribeirão Preto. ‘We isolated the intestinal contents of these organisms and cultivated in specific carbon sources, such as xylan, xylose, CMC, etc. to select microorganisms present there who had the features of interest. At this time, the Pseudozyma stood out’, says Oliveira.

The researchers decided to sequence the genome of yeast due to its biotechnological potential and the unique environment in which it was isolated. This step included the work of computational biology of CTBE researcher, Diego Mauricio Riaño-Pachón It was up to him to assemble the genome of 20 million base pairs of the species sequenced in a US laboratory, from packages of 100 pairs. He also identified and gathered the continuous sequences of genes within the long encoded chain and realized what information was present in the genome. This made it possible to compare the material obtained with the content of international databases, in the search for characteristics typical of species of this genus. ‘By comparing the genes, we found sufficient differences to consider it a new species’, adds Riaño-Pachón.

The Knowledge of genome of the new Pseudozyma is essential for the development of genetic changes in microorganisms able to improve their biotechnological characteristics. Riaño-Pachón says it’s not worth replacing genes in one species without knowing how these operate and interrelate with others. Thus, the CNPEM team now performs other analyzes, such as RNA-seq, where the aPseudozyma is placed to grow in carbon sources, such as xylose and xylan, with the intention of identifying the most important genes for the promotion of the enzymatc activity genes, allowing the expression of these in industrial yeasts.

The sequence of the P. brasiliensis is already available in the GenBank, managed by National Center for Biotechnology Information (NCBI). Following, the description of the genome was published in the journal Genome Announcements, as well the announcement of a new species in a taxonomy magazine.

Participated in the studies the CTBE/CNPEM researchers Gustavo Goldman, Juliana Velasco de C. Oliveira and Diego Mauricio Riaño-Pachón. The research received financial support from the Foundation for Research Support of the State of São Paulo (FAPESP), of the National Council for Scientific and Technological Development (CNPq) and Vale company.

.

Computational biology

The genome assembly, the structural and functional annotation and the phylogenetic analysis of the new Pseudozyma only took about six months to be completed, thanks to the infrastructure of high performance computing available at CNPEM. Riaño-Pachón explains that the lack of this type of installation, along with the lack of skilled labor is the biggest current obstacle for the expansion of the works in bioinformatics and other areas related to computational biology in Brazil.

“Until recently, one of the major bottlenecks of biotechnology was in the production of scientific data. Currently, such production is fast and inexpensive. The problem now is how you handle large volumes of data in a short period of time’, says Riaño-Pachón.